The technical objective pursued with the present invention is, precisely, the development of formulations capable of enhancing the immune response to antigens administered through mucosal routes, minimizing the number of components in the formulation. The enhancing activity is supported by the interaction between particles at the mucosal level, generating systemic as well as mucosal immunity. Furthermore, the development of combined vaccines to the mucosal route taking as a central antigen the HBsAg, increased the immune response to one or more of coadministered antigens. The obvious advantage is the elimination of all other element or compounds different from the antigen of interest and the use of a different route. We consider that this is the basis or nucleus to develop combined vaccines for a mucosal use.
HBcAg is an extremely immunogenic antigen during the Hepatitis B Virus (HBV) infection or after immunization. In many HBV chronic patients, this is the only antigen capable of inducing an immune response. It can even induce an immune response in mice in nanogram quantities. Recently, a few structural studies have demonstrated some important characteristics explaining its potent immunogenicity. HBcAg specifically binds membrane immunoglobulin receptors in a large number of resting B cells in mice, which is sufficient to induce costimulatory B7-1 and B7-2 molecules. In this way, non-sensitised B cells, specific for HBcAg can uptake, process and present HBcAg peptides to naive T cells in vivo and to T cells hybridoma in vitro, approximately 105 times more efficiently than macrophages and dendritic cells. This structure-function relationship explains the great immunogenicity of HBcAg (Milich, D. R. et al. 1997 Proc. Natl. Acad. Sci USA December 23; 94(26): 14648-53). Serologic and biochemical studies indicate that the resolution of HBV acute infection occurs in the context of an efficient cell-mediated immune response, while the chronic infection is characterized by a poor and undetectable cell-mediated immune response and a “relatively efficient” humoral response.
The humoral immunity and the cell-mediated immunity are regulated by different groups of helper T cells. Factors influencing the induction in mice of a Th1 or Th2 response to the HBV antigens (HBcAg/HBeAg) revealed that this balance was influenced (1) by the antigen structure (HBcAg is a particulated structure and HBeAg is not; (2) the major histocompatibility complex (MHC) of the host and the T cell antigens which are recognized; (3) the cross regulation between Th1 and Th2 cells; (4) the T cell tolerance, which is more complete for Th1 than for Th2 cells; (5) the activity of secreted HBeAg that preferentially delete Th1 cells (6) the treatment with cytokines, used to modulate in vivo the response toward Th1 or Th2 cells. This balance Th1/Th2 is relevant to the acute or chronic course of the HBV infection. Th2 cells preferentially evade the induction of tolerance compared with Th1. As HBeAg acts as a tolerogen during HBV vertical transmission, deleting Th1 cells, the predominance of Th2 specific cells for HBeAg could influence in the initiation and maintenance of a chronic carrier state. In this case the cytokine therapy endowed to modulate the response towards Th1, could be benefitious in the treatment of HBV chronic infection (Milich, D. R. 1997 J. Viral. Hepat.; 4 Suppl 2: 48-59).
The effect of HBeAg circulation on HBcAg Th1 specific T cells was examined by transferring HBeAg/HBcAg specific T cells to double (HBeAg and HBcAg) transgenic mice. The presence of serum HBeAg eliminated the Th1 mediated response against HBcAg and changed the balance to the Th2 phenotype. This result suggest that, in the context of the hepatitis B infection, circulating HBeAg has the potential to preferentially eliminate inflammatory specific Th1 cells needed for viral clearance, promoting the persistency of HBV (Milich-DR et al. 1998 J-Immunol. February 15; 160(4): 2013-21).
It is known that antibodies against HBcAg are present since the beginning of the infection and reach high concentrations in sera of HBV chronically infected patients, but these antibodies are not protective. Antibodies passively transmitted to newborn children by chronic carrier mothers, do not protect children of infection. (Beasley et al. 1977. American Journal of Epidemiology 105: 914-918). However, it has been demonstrated that immunizing chimps with HBcAg partially or completely protected them from HBV infection (Iwarson, S. et al. 1985 Gastroenterology 88: 763-767; Murray, K. et al. 1987 Journal of Medical Virology 23: 101-107). In Iwarson's study, three chimps were completely protected. After challenge with HBV, antibody levels against HBcAg and HBeAg increased but only one chimp seroconverted against HBsAg. In Murray's study, 2 out of 4 immunized chimps showed a low level of viral replication after challenge, HBsAg was detectable in sera for 2 or 3 weeks, and after that they developed an anti-HBsAg antibody response. It was hypothesized that the incomplete protection could be due to the low immune response in vaccinated animals without adjuvant.
After immunizing with woodchucks hepatitis core antigen (WHcAg) in Freund Complete Adjuvant (ACF), it was possible to protect woodchucks from challenge with the virus (WHV) without signs of infection detectable antibodies against the surface protein (WHsAg). Although the hypothesis that T helper anti-nucleocapsid immune response could enhance undetectable antibodies against the surface antigen can not be discarded, the cytotoxic activity was considered as the main responsible of protection (Roos, S. et al. 1989 J. Gen. Virol. 70, 2087-2095). In a second study using woodchucks the role of HBcAg and WHcAg in protection was determined as well as the possible mechanism. Animals were immunized with WHcAg and HBcAg and afterwards challenged using a high dose of WHV. In this experiment it was found that WHcAg is a protecting antigen there is a cross protection because 4 out of 6 woodchucks immunized with HBcAg were protected from the challenge. Both antigens generated a high antibody titer with a cross reactivity lower than 1%, confirming previous reports of protection using internal hepatitis B virus antigens. Since dominant B epitopes of both antigens do not appear to be conserved, this result also demonstrated that antibodies directed against core antigens are not important for protection. Woodchucks immunized with WHcAg/HBcAg reacted with a rapid response of serum antibodies against surface proteins after challenge with WHV, indicating an increased helper T cell response as a potential mechanism of protection after immunization with an internal antigen of HBV/WHV. (Schodel-F et al. Vaccine. 1993; 11(6): 624-8) Transfection of established cell lines from BALB/C mice hepatocytes with dimeric HBV DNA (ML lines) resulted in the expression of HBV antigens in these lines. The adoptive transference of spleen cells of BALB/c mice immunized with ML-1.1 cells expressing HBsAg as well as HBcAg, caused a regression of tumours cells expressing the corresponding antigens in athymic mice. Furthermore, the transfer of spleen cells of BALB/c mice immunized with HBsAg or HBcAg also caused tumoral regression. These results demonstrated that surface and nucleocapsid antigens could induce immunity capable of rejecting the hepatocellular carcinoma in vivo (Chen, S. H. et al. 1993 Cancer-Res. October 1; 53(19): 4648-51).
Therapeutic vaccines based on specific nucleocapsid epitopes for human HLA are being assayed in phase II/III studies (Liaw, Y. F. 1997 J. Gastroenterol. Hepatol. October, 12 (9-10): S227-35).
HBcAg has been demonstrated to be a very good carrier. HBcAg is a highly immunogenic antigen in human and animal models. HBcAg directly activates B cells and generates strong T cell responses. Furthermore, the efficient processing and presentation of HBcAg by the antigen presenting cells makes it an ideal carrier molecule. Hence a large number of epitopes have been chemically linked or genetically fused to the HBcAg molecule to successfully increase their immunogenicity. Bacterial expression vectors have been designed to enable the insertion of heterologous B cell epitopes in different positions inside the particles of HBcAg and the efficient purification of hybrid particles.
Positional studies of B cell epitopes demonstrated that internal insertions by the amino acid 80 continue to be immunodominant, permitting an increase in the production of antibodies as compared to other fusion proteins. Immunogenicity studies have been performed with experimental challenge in different systems. For example, a peptide from Plasmodium berghei Circunsporozoite was inserted in this site and the purified hybrid particle HBcAg/CS was highly immunogenic and protected 100% of challenged mice against malaria. Aimed to the development of oral vaccines, attenuated avirulent Salmonella strains have been used to introduce genes coding for hybrid particles of HBcAg (Milich, D. R. et al. 1995 Ann. N.Y. Acad. Sci. May 31; 754: 187-201).
In conclusion, apart from the relationship between HBcAg and protection, total or partially evidenced in chimps or indirectly referred by the experiments with WHcAg, this protein has a number of properties that makes it unique. HBcAg behaves as a T dependent as well as a T independent antigen (Milich, D. R. et al. 1986 Science 234, 1398-1401), it is very immunogenic, even without the help of adjuvants and its inoculation preferentially sensitises Th1 cells (Milich, D. R. et al. 1997, J. Virol. 71, 2192-2201). HBcAg is a very efficient carrier protein (Schödel, F. et al. 1992 J. Virol. 66: 106-114; Milich-DR et al. 1995 Ann-N-Y-Acad-Sci. May 31; 754: 187-20) and Th HBcAg specific cells mediate the antibody response against HBcAg as well as anti-HBsAg (Milich, D. R. et al. 1987 Nature (London) 329: 547-549). These immunologic characteristics are unique for the particulated HBcAg and do not apply to the non-particulated form of the antigen, the HBeAg (Milich, D. R. et al. 1997 Proc. Natl. Acad. Sci USA December 23; 94(26): 14648-53).